重组GIP蛋白的原核优化表达及其生物活性的研究

Optimized Prokaryotic Expression and Bioactivities of The Recombinant Human Glucose-dependent Insulinotropic Polypeptide

葡萄糖依赖性促胰岛素多肽或抑胃肽(glucose-dependentinsulinotropicpolypeptideorgastricinhibitorypeptide,GIP)是由42个氨基酸组成的胃肠调节肽,在高血糖背景下能够刺激胰岛素释放,能够抑制胃酸分泌、促进神经细胞增生,具有广泛的临床应用价值.化学提取或人工合成GIP,成本过高,不宜规模化生产,故应用基因工程技术研制重组人GIP(rhGIP)并探讨其生物活性有积极的现实意义.人工合成具有大肠杆菌偏爱密码子的编码GIP成熟肽的cDNA序列,利用pET32a(+)系统进行原核表达;在小规模发酵条件下,进行优化诱导表达和目的蛋白的亲和纯化;通过检测SD大鼠胃酸分泌和血糖浓度,对纯化后的rhGIP进行生理活性研究;通过形态学观察和培养基中NO含量测定,检测rhGIP对PC12细胞NO自由基生成量的影响;应用Aβ25-35加入培养基造成PC12细胞神经损伤模型,分别以高、中、低剂量rhGIP作用于此模型,通过MTT(2-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazoliumbromide)法测定PC12细胞的活性.结果显示,成功克隆了人GIP基因,诱导表达的rhGIP占细胞总蛋白质的35%,部分可溶,部分以包涵体形式存在.经过诱导表达的重组蛋白质分子质量约为26ku,与理论值相符.纯化后的rhGIP具有免疫活性.优化诱导表达条件为表达菌生长密度A600值0.50,IPTG浓度0.5mmol/L,温度37℃,诱导表达时间4h.裂解上清液经固定化金属亲和层析一步法层析后,表达的水溶性rhGIP融合蛋白的最后得率为1.2mg/L菌液,纯度为85%.纯化后的rhGIP能够使SD大鼠胃液pH值增高,其抑制胃酸分泌作用与生理盐水对照组比较差异有显著性(P<0.05),而rhGIP组和标准品GIP组比较差异无显著性.在高血糖背景下,注射rhGIP15min后,大鼠血浆血糖浓度较基础血糖显著降低(P<0.05),30min时与单独注射葡萄糖的模型对照组比较,差异无显著性,而rhGIP组和标准品GIP组其差异不显著.在rhGIP对神经细胞的营养和对PC12细胞免受神经损伤和缺氧损伤影响的研究中发现,用rhGIP培养PC12细胞32h后,rhGIP组NO含量极显著低于正常对照组(P<0.01),细胞存活较多,神经突起延伸较好,中、高剂量rhGIP组均较神经损伤和缺氧损伤组活性显著升高(P<0.05),且细胞活性呈剂量依赖关系,rhGIP组与标准品GIP组差异不显著,与正常对照组亦无显著差异.研究结果表明,已得到高效表达的rhGIP融合蛋白,该蛋白质具有免疫活性,具有抑制胃酸分泌和降低大鼠血浆血糖浓度的生理活性,并且对神经细胞有营养和保护作用.

GIP, the acronym from gastric inhibitory peptide or glucose-dependent insulinotropic polypeptide, which is a kind of gastrointestinal regulatory peptide composed of 42 amino acids, plays an important role in stimulating insulin releasing in the pancreas, inhibiting gastric acid secretion in the stomach, as well as inducing progenitor cell proliferation in the brain. The application of GIP is promising in clinic. It is difficult and costly to get GIP with chemical extraction method, and it can＇t be produced cosmically, so it is valuable and significant to produce recombinant GIP by genetics and test its bioactivity. The cDNA sequence coding for human GIP mature peptide and composing of preferred codons of E. coli. was synthesized, and was expressed in prokaryotic expression pET32a （＋） system. The recombinant E. coli was fermented in a small scale and the expression condition was optimized. The expressed recombinant human GIP （rhGIP） was purified by affinity method. The bioactivity of the purified rhGIP was tested through its effect on inhibiting gastric acid secretion and decreasing blood glucose concentration in SD rats. The effect on PC12 cell producing NO free radical was assessed through morphology observation and the NO concentration test. A PC12 cell injury model was constructed by adding Aβ25-35 into the culture medium, different dose of rhGIP was added into this model, and the activity of PC12 was tested by MTT （2-（4,5-dimethylthia-zol-2-yl）-2,5-diphenyltetrazolium bromide） staining. The results suggest that hGIP gene is cloned successfully, the recombinant protein constitutes 35% of the total protein of the cells, some of which was soluble, while the others exist as inclusion body. The relative molecular mass of the recombinant protein is 26 ku as expected. The purified rhGIP showed immunoreactivity. The optimal condition for inducing to expression is with the cell concentration of 0.5 in A600 value, the IPTG concentration of 0.5 mmol/L, under 37℃ and induced for 4 h. The cell lysis supematant was purified with immobilized metal affinity chromatograph. The production of soluble rhGIP is about 1.2 mg/L culture medium, and the purity is 85%. Purified rhGIP significantly inhibited the secretion of gastric acid and raised the pH value comparing with NC group in SD rats （P 〈 0.05）. There was no significant difference between the rhGIP and standard GIP group （P 〉 0.05）. After injection of rhGIP for 15 min, the concentration of blood glucose in plasma decreased remarkably comparing with control group in a high blood glucose environment （P 〈 0.05）. In the later 30 min, there was no remarkable difference （P 〉 0.05）. Also, there was no difference between rhGIP and standard GIP group （P 〉 0.05）.In the research to test the effect of rhGIP on cell nutrition and PC12 cell to avoid neural injury and anoxic injury, it was found that the level of NO was significantly decreased （P 〈 0.01）, the survival rate of the PC12 cells was increased, and neurite extending was better. The activity of the high and medium rhGIP concentration group is higher than that of neural injury group and anoxic injury group （P 〈 0.05）, and the activity is increased as dose-dependence. The differences among rhGIP, GIP and NC group is not remarkable （P 〉 0.05）. The results showed that the rhGIP has been successfully expressed and purified and showed obvious immunoactivity. The recombinant product was shown to have a wide spectrum of biological activity, such as inhibiting gastric acid secretion in the rats stomach, decreasing the concentration of blood sugar in plasma, as well as nutrition and protection to neural cells.